G protein-coupled receptors (GPCRs) are critical eukaryotic signal transduction gatekeepers and represent the largest protein family in the human proteome, with more than 800 members. In recent years, X-ray crystallography has yielded many G protein-coupled receptor structures but the mechanism of allosteric signaling remains unknown. X-ray crystallography of GPCRs requires multiple forms of receptor stabilization that, subsequently, limit conformational dynamics. RESEARCH: Directed evolution of GPCRs enables milligram quantities of functional, isotopically-labeled protein to be produced from prokaryotic expression systems. This technology opens the field for NMR studies of GPCRs with various ligands and in complex with G proteins and arrestins. Focusing on the neurotensin receptor, the PI will use NMR spectroscopy to probe the structure and conformational dynamics of GPCR activation in solution. TRAINING: The proposed training in Professor Wagner's laboratory at Harvard Medical School will solidify the PI's comprehension of NMR theory and implementation. In addition to advanced NMR techniques, the PI will acquire a thorough understanding of membrane protein biochemistry, nanodiscs, and directed evolution. Together, these techniques will empower the PI to establish an independent, NIH-funded structural biology research program. ENVIRONMENT: Professor Gerhard Wagner's laboratory at Harvard University Medical School presents the optimal environment for this project. His work has been integral to the development of multidimensional NMR experiments for biomolecule characterization; in particular, Dr. Wagner is a leader in data collection and analysis of large macromolecules. Harvard Medical School has nearly 8,000 faculty and 17 affiliated facilities. At the core of the Medical School are its educational and research programs. The Medical School has nine departments in basic and social science disciplines. The wider Boston/Cambridge area contains numerous research laboratories including MIT, Whitehead, and Broad Institutes with interests in structural biology, biochemistry, molecular biology, chemistry and physics, with scientists interested in the general themes related to this proposal. IMPACT ON PUBLIC HEALTH: GPCR-mediated signaling pathways have been related to numerous human diseases, and GPCRs are the targets of an estimated 30-40% of all drugs currently on the market. In view of their fundamental roles in health and disease, a detailed understanding of GPCR structure and function is of value to the basic science community interested in cell signaling and molecular recognition, as well as to the applied science community interested in drug discovery.
GPCR-mediated signaling pathways are involved in countless human diseases, and GPCRs are the target of nearly 40% of all drugs currently on the market. A detailed understanding of GPCR structure and function is critical to the applied science community interested in drug discovery.
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| Kleist, Andrew B; Getschman, Anthony E; Ziarek, Joshua J et al. (2016) New paradigms in chemokine receptor signal transduction: Moving beyond the two-site model. Biochem Pharmacol 114:53-68 |
